PROJECT ABSTRACT The purpose of this research project is to mature human pluripotent stem cell-derived cardiomyocytes (hiPSC- CMs) via transcriptional programming, such that they display adult-like electrophysiological properties. hPSC- CM applications for cell therapy and disease modeling are limited due to the cells? lack of resemblance structurally and functionally to their adult counterparts. The development of arrhythmias has been a major problem after transplantation of hPSC-CMs into non-human primates and thus presents a major obstacle in the translation of hPSC-CMs to the clinic. I hypothesize that the immature state of hPSC-CMs is a major contributor to the graft-induced arrhythmias, and transcriptional programming of these cells will be an effective system to enhance the maturation state of hPSC-CMs in vitro for various applications. The specific aims for this project are: 1) to identify a core set of cardiac transcriptional regulators that can be used to combinatorially program hiPSC-CM electrical maturation and induce adult-like cardiac electrophysiological properties, and 2) to elucidate the chromatin structural mechanisms by which maturation transcriptional regulators coordinate differential gene expression. We have identified candidate transcriptional regulators by studying human cardiomyocyte maturation in vivo after transplantation. We will activate these regulators using CRISPR activation (CRISPRa) to test their sufficiency in generating electrically mature cardiomyocytes. Maturation will be measured by single cell RNA-sequencing analyses and follow-up assessment of electrophysiological properties. Next, chromatin immunoprecipitation-sequencing will be performed to identify candidate transcription factor target genomic binding sites. ATAC-sequencing will be used to assess regulation of genome accessibility in programmed hiPSC- CMs and to characterize the epigenetic mechanisms by which top candidate factors fully and/or partially program maturation gene expression. This project presents a new approach to mature hiPSC-CMs by using high throughput and combinatorial CRISPR-based screening and will provide characterization of chromatin structural features regulating cardiomyocyte maturation. Through generating more mature hiPSC-CMs, these programmed cardiomyocytes can be applied as cell therapy to remuscularize damaged heart tissue in patients after myocardial infarction, while bypassing graft-induced arrhythmias. This project will build on the applicant?s research training and skillsets and will serve to foster the applicant?s path towards becoming an independent investigator.